Abstract: Asthma is a lung disease that causes constriction of the airways in response to triggers such as pollen, mold, viral infection, or even cold air. In severe cases, the obstruction f the airways in asthma can be fatal. Asthma particularly affects a large number of children and the disease is growing in prevalence worldwide. In the majority of asthma patients, chronic infiltration of the lungs with inflammatory cells is present. These inflammatory cells have been shown to make an important contribution to disease pathology, yet current treatments for the disease are inadequate. Considerable progress has been made in defining the specific types of inflammatory cells that accumulate in the lungs of asthma patients. A large number of studies have been directed at testing the roles of these individual cell types by genetic ablation or the inhibition of particular cell-associated molecules by pharmacological agents, followed by measurement of the effects on lung function and inflammation. However, while this experimental approach has identified many of the key pieces that make up the asthma puzzle, it has failed to provide a picture of how these pieces fit together to cause disease. Indeed, little is known about the cell-cell interactions in vivo that lead to early allergic sensitization or subsequent chronic asthma. This study proposes to apply cutting-edge microscopy techniques to the characterization of cellular interactions in asthma using mouse models. The first step of this study will be to identify methods to visualize inflammatory cells in the lungs by two-photon microscopy. The particular cell types of interest will be mast cells, basophils, eosinophils, neutrophils, macrophages, monocytes, dendritic cells, T cells, and B cells. These cells will be specifically visualized by the expression of fluorescent proteins in a lineage-specific fashion, or by labeling with fluorescent dyes or antibodies. In several cases, this study will be able to draw upon existing lines of transgenic mice for this purpose. The second step of this study will then be to characterize the interactions among these cells by two-photon microscopy. The particular cell types that interact with each other will first be identified, and then the frequency and duration of these contacts will be evaluated. The nature of these contacts will then be probed by genetic and pharmacological perturbations. The third step in this study will be to identify the locations of these cellular interactions within the lung and define the features of the tissue microenvironment that promote these interactions. A particular area of emphasis will be the interaction of inflammatory cells with stromal components of the lung, including epithelial cells and smooth muscle cells. Taken together, this study will provide new insights into how inflammatory cells communicate with each other and lung-resident cells leading to asthma pathogenesis. These findings are likely to provide a new paradigm for the understanding of asthma, which may ultimately lead to novel therapeutic interventions. Public Health Relevance: This study aims to improve our understanding of how inflammation contributes to asthma. The overall goal of this study is to determine which inflammatory cells in the lungs interact with each other and how they communicate to cause the disease. The results of this work may provide a foundation for new types of asthma therapeutics that target these cellular interactions.